The overall objective of the proposed research project is to use a defined in vitro experimental system to study the molecular basis of phenotypic variation and explore its relationship to epigenetic control of gene expression, somatic mutation, and tumor progression in hepatoma cells. Also, the connection between expression of embryonic and adult liver antigens (both membrane-bound and secreted) and malignant potential will be explored. The specific example of this phenotypic variation, which I have studied extensively, is the clonal variation in albumin synthesis in cultured hepatoma cells. Initially, the clonal variation in rate of albumin synthesis will be thoroughly characterized on a single cell basis in clonal hepatoma cell lines using both qualitative and quantitative techniques (immunoperoxidase staining, immunofluorescence, flow cytofluorometry) and the genetic, epigenetic, and environmental influences evaluated. Antisera against cell-type-specific surface antigens of adult and fetal hepatocytes will be prepared as well as assay systems for alpha-fetoprotein, fibrinogen, and gamma-glutamyl transpeptidase. Using the techniques for quantitating at the single cell level, a coordinated study of control of expression of these specific products in hepatoma cells will be undertaken to establish if a similar clonal variation is observed for different functions in the same cells, determine their rate of phenotypic variation (RPV), establish if the variation follows the same geometric progression as seen for albumin synthesis and if there is a correlation between the RPV of any of these specific liver functions and malignant potential. Also, using an albumin cDNA probe, the relationship between rate of albumin synthesis and albumin gene frequency will be studied.